Effects of Suoquan Yishen Formula on Mice with Diabetic Nephropathy (DN)

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1. Hainan Medical College, Haikou 571199, China; 2.Guizhou University of Traditional Chinese Medicine, Guiyang 550025, China; 3. Capital Medical University, Beijing 100054, China

Abstract [Objectives] The purpose of this paper was to study the effects of Suoquan Yishen formula on mice with diabetic nephropathy (DN). [Methods] The mice were randomly divided into normal group, model group, irbesartan group, and high and low-dose Suoquan Yishen formula groups. The blood glucose level, BUN, Scr excretion and GSH activity and CAT activity were detected. [Results] The blood glucose levels of mice in the irbesartan group and high and low-dose Suoquan Yishen formula groups decreased. The BUN, PRO and Scr levels were higher and the GSH and CAT activity was lower in the model group compared with the normal group. After administration of Suoquan Yishen formula, the BUN, PRO and Scr levels of the DN mice decreased significantly, and the GSH and CAT activity increased significantly. [Conclusions] Suoquan Yishen formula can reduce blood glucose level, reduce PRO, BUN and Scr levels, and increase CAT and GSH activity in DN mice, thus protecting the ND mice.

Key words Diabetic nephropathy, Suoquan Yishen formula, Physiological indicator

1 Introduction

Suoquan Yishen formula is made from Suoquan pill combined with the characteristics of regional diseases in Hainan. It consists of Fructus Alpiniae Oxyphyllae, Radix Linderae, Radix Paeoniae Rubra, Radix Trichosanthis, Rhizoma Atractylodis, Radix Salviae Miltiorrhizae and poria. The prescription is composed of monarch, minister, adjuvant and guide, and is closely related to pathogenesis, characterized by rigorous compatibility and complement to each other, with functions of warmly invigorating kidney yang, resolving phlegm and invigorating blood circulation. It is mainly used to treat diabetic nephropathy (DN) of kidney deficiency and stasis-phlegm type. It has been applied in clinics for many years, and obtained the invention patent authorization. Suoquan Yishen formula has been proved to have definite effect on DN of kidney yang deficiency. However, the mechanism of the protective effect of Suoquan Yishen formula on diabetic mice is not clear. Based on the establishment of mouse model of DN, the blood glucose level, urinary BUN, PRO and Scr levels, and serum GSH and CAT activity of the mice were detected to explore the protective effect of Suoquan Yishen formula on diabetic mice.

2 Materials and methods

2.1Materials

2.1.1Animal. All mice were purchased from the Model Animal Research Center of Nanjing University. After one week of routine adaptive feeding, the mice without adverse reactions were selected for the experiment.

2.1.2Drugs. Decoction of Suoquan Yishen formula (Tongrentang) was prepared by the authors. Metformin hydrochloride tablets were produced by Sanofi (Hangzhou) Pharmaceutical Co., Ltd.

2.1.3Reagents and instruments. The reagents and instruments used included catalase (CAT) kit (A007-2, Jiancheng, China), glutathione (GSH) kit (A006-2, Jiancheng, China), blood glucose meter (ACCU-CHEK Performa, Germany), blood glucose test stripes (ACCU-CHEK Performa, Germany) and full-wavelength microplate reader (BioDeK Instruments Inc, USA).

2.2Methods

2.2.1Grouping and administration. Ten DB-/DB- mice were selected as normal group. Forty db-/db- mice were evenly and randomly divided into four groups: model group, irbesartan group, high-dose Suoquan Yishen formula group and low-dose Suoquan Yishen formula group. The rats in the drug administration groups were given with corresponding drugs at a dose of 0.2 mL (once a day), and those in the normal group and model group were given with equal-amount normal saline. The administration lasted for 4 weeks.

2.2.2Specimen collection. The tails of the rats were cut once a week for collecting blood. The blood glucose level was determined. After 4 weeks of intragastric administration, 24-h urine of each of the rats was collected; and their blood was collected from the orbits. The blood samples were centrifuged to collect the serum for indicator determination.

2.2.3Indicators determined. For observation of daily state, the mental activity, hair, feed intake and water intake, as well as body mass and mortality of the rats were observed and recorded.

For determination of blood glucose level, blood of the rats was collected by cutting their tails. The blood glucose level was measured with a blood glucose level and test stripes.

Before slaughter, 24-h urine of the each of the rats was collected. According to the instructions of the kits, Scr, BUN and PRO levels were determined.

Eyeball blood of each of the rats was collected. The blood samples were centrifuged to collect the serum. According to the instructions of the kits, the CAT and GSH activity was determined.

3 Results and analysis

3.1StateCompared with the normal group, the ND mice showed higher drink intake, more urine, loose hair, weak spirits, and significantly increased body weight (P<0.01). Compared with the model group, the general status of the rats in the irbesartan group and Suoquan Yishen formula groups was improved, but their body weight did not change significantly (Table 1).

GroupDoseg/kgBody mass∥gBeforeadministrationAfter 2 weeks ofadministrationAfter 4 weeks ofadministrationNormal-18.32±0.8523.98±1.3119.29±1.83Model-20.84±1.2242.69±2.50∗52.50±2.43∗Irbesartan0.01020.73±1.9346.89±1.79∗54.61±2.90∗Suoquan Yishen 0.01021.86±1.5550.06±1.91∗53.36±1.38∗formula0.00522.06±1.6349.22±2.09∗51.71±2.91∗

Note: Compared with the normal group,*P<0.01.

3.2BloodsugarIn comparison with the normal group, the blood glucose level of the model group significantly increased (P<0.05). Compared to the model group, the blood glucose levels of the irbesartan group and Suoquan Yishen formula groups began to decrease significantly from the 4th week (P<0.05) (Table 2).

GroupDoseg/kgBody mass∥gBeforeadministrationAfter 2 weeks ofadministrationAfter 4 weeks ofadministrationNormal-8.52±1.328.36±0.946.54±1.94Model-27.03±1.61#28.75±2.44#31.67±1.11#Irbesartan0.01027.10±1.3327.57±1.6825.90±1.97∗Suoquan Yishen0.01028.01±1.0627.08±1.5225.35±1.77∗formula0.00527.67±1.9027.40±1.0726.05±0.98∗

Note: Compared with the model group,*P<0.05; compared with the normal group,#P<0.05.

3.3RenalfunctionindicatorsIn comparison with the normal group, the 24-h PRO, Scr and BUN levels of the model group significantly increased (P<0.05). Compared to the model group, after 4 weeks of administration, the Scr and BUN levels of the irbesartan group and the PRO, Scr and BUN levels of the Suoquan Yishen formula groups decreased significantly (P<0.05) (Table 3).

GroupDoseg/kgPROmg/24 hScrmg/24 hBUNmg/24 hNormal-0.037±0.0130.11±0.070.40±0.21Model-1.920±0.15#3.01±0.08#60.56±1.18#Irbesartan0.0102.620±0.710.77±0.02∗9.51±1.01∗Suoquan Yishen0.0100.410±0.13∗0.36±0.13∗8.97±2.10∗formula0.0050.770±0.32∗0.41±0.01∗8.75±1.51∗

Note: Compared with the model group,*P<0.05; compared with the normal group,#P<0.05.

3.4OxidativestressCompared with the normal group, the serum GSH and CAT activity of rats in the model group significantly decreased (P<0.05). In comparison to the model group, after 4 weeks of administration, the serum GSH and CAT activity of rats in the irbesartan group and Suoquan Yishen formula groups significantly increased (P<0.05) (Table 4).

GroupDose∥g/kgGSH∥μmol/LCAT∥U/mLNormal-4.73±3.4227.12±3.51Model-2.49±0.75#23.81±3.06#Irbesartan0.0103.98±0.73∗41.48±2.19∗Suoquan Yishen formula0.0103.51±2.26∗30.22±1.11∗0.0052.79±1.2526.95±5.46

Note: Compared with the model group,*P<0.05; compared with the normal group,#P<0.05.

4 Discussion

DN mainly has symptoms such as presence of protein in the urine, high blood glucose level, polydipsia, polyphagia, polyuria,etc. It is a disease with complex and diverse development mechanisms. Factors such as renal hemodynamics, exosomes, inflammation and miRNA all can cause diseases[1-9].DN is similar to the diagnostic concepts of edema, gonorrhea, dysuria and vomiting in traditional Chinese medicine. They all showed clinical symptoms such as dribble urination, changed urination habit, changed urination frequency and partial body edema. There are many types of syndromes, and the most common type is kidney yang deficiency. Kidney yang deficiency, caused by deficiency in kidney yang, failure to warm up and failure in qi transformation, is a type of deficiency syndrome manifested by intolerance of cold and cold limbs (especially below the waist), fatigue, long voidings of clear urine, increased nocturnal urine, light-colored tongue with white coating and weak pulses. The pathogenesis is mainly due to failure of spleen-kidney, lucid yang failing to rise and turbid yin failing to drop, with many symptoms. Therefore, the main therapy is to warm the kidney yang and to restore energy.

Suoquan Yishen formula is composed of seven herbs. Among them, Fructus Alpiniae Oxyphyllae has the effects of warming the kidney qi and warming the kidney yang. It promotes blood circulation in the lower limbs and is good at securing essence and reducing urination. Fructus Alpiniae Oxyphyllae is used to treat frequent urination, enuresis, gonorrhea, nocturnal emission and premature ejaculation caused by deficiency in kidney (qi) yang. Radix Linderae has the effects of promoting the circulation of qi, relieving pain, warming the kidney and dispersing cold. The compatibility of the two herbs can warm lower limbs, disperse cold, tonify spleen and kidney and reduce urination, and they are both monarchs in the prescription. Radix Paeoniae Rubra can clear heat, cool blood, remove blood stasis and relieve pain. Radix Salviae Miltiorrhizae can remove blood stasis, relieve pain, promote blood circulation, and stimulate menstrual flow. The two herbs above must be accompanies by each other. Rhizoma Atractylodis can dry dampness, tonify spleen, expel wind, remove cold and improve eyesight. Radix Paeoniae Rubra, Radix Salviae Miltiorrhizae and Rhizoma Atractylodis are ministerial drugs, and they assist the monarchs together to strengthen the functions of promoting blood circulation, removing blood stasis, relieving pain, drying dampness and tonifying spleen. Radix Trichosanthis, as an adjuvant, can clear heat, engender fluid, diminish swelling and expel pus. Poria, as an envoy drug, can benefit the spleen and stomach and reduce phlegm. The drugs complement each other, and have good clinical application effect for DN of stasis-phlegm type.

Long-term glucose metabolism disorder is the underlying cause of DN. In this study, the blood glucose levels of the Suoquan Yishen formula groups significantly declined compared with the model group, and the decline in blood glucose level was positively correlated with the dose, consistent with many studies on blood glucose reduction for DN[10-12].DN is a process of slowly progressive kidney damage. The main clinical indicators are glomerular filtration rate and Scr, BUN and PRO levels[13-14]. In this study, by measuring the effects of drugs on the expression of PRO, Scr and BUN in DN mice, it was confirmed that Suoquan Yishen formula can downregulate the expression of the three substances, and the inhibitory effect of high-dose Suoquan Yishen formula was greater, with better protective effect on kidney injury. There is a close relationship between oxidative stress level and diabetes. The reduction of antioxidant capacity is another important cause of diabetes complications[15-17].Masudaetal.[18]found that T2DM patients have significant oxidative stress in their kidneys, and oxidation can cause damage to the podocytes in basement membrane. Under the action of high glucose level, multiple metabolic pathways can cause oxidative stress, and reactive oxygen species can also play a role. As the number increases, it affects activation pathway, leading to further increase in the number. The cycle repeats, gradually aggravating. The vicious circle makes the kidney cells constantly damaged and their functions affected, and thus, damage occurs[19].CAT and GSH are indicators of antioxidant stress. The greater the activity, the stronger the antioxidant effect[20]. In this study, the effects of drugs on serum CAT and GSH activity in DN mice were determined, and the results show that Suoquan Yishen formula can increase the activity of CAT and GSH, and high-dose Suoquan Yishen formula has better effect, and it can enhance the body’s antioxidant capacity.

In summary, the results of this study show that Suoquan Yishen formula can reduce blood glucose level, reduce PRO, BUN and Scr levels, and increase CAT and GSH activity in DN mice, thus protecting the DN mice.